Conservation of binding site specificity of three yeast DNA binding proteins

AbstractSequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited

Comparison of targeted metagenomics and IS-Pro methods for analysing the lung microbiome

BACKGROUND : Targeted metagenomics and IS-Pro method are two of the many methods that have been used to study the microbiome. The two methods target different regions of the 16 S rRNA gene. The aim of this study was to compare targeted metagenomics and IS-Pro methods for the ability to discern the microbial composition of the lung microbiome of COPD patients. METHODS : Spontaneously expectorated sputum specimens were collected from COPD patients. Bacterial DNA was extracted and used for targeted metagenomics and IS-Pro method. The analysis was performed using QIIME2 (targeted metagenomics) and IS-Pro software (IS-Pro method). Additionally, a laboratory cost per isolate and time analysis was performed for each method. RESULTS : Statistically significant differences were observed in alpha diversity when targeted metagenomics and ISPro methods’ data were compared using the Shannon diversity measure (p-value = 0.0006) but not with the Simpson diversity measure (p-value = 0.84). Distinct clusters with no overlap between the two technologies were observed for beta diversity. Targeted metagenomics had a lower relative abundance of phyla, such as the Proteobacteria, and higher relative abundance of phyla, such as Firmicutes when compared to the IS-Pro method. Haemophilus, Prevotella and Streptococcus were most prevalent genera across both methods. Targeted metagenomics classified 23 % (144/631) of OTUs to a species level, whereas IS-Pro method classified 86 % (55/64) of OTUs to a species level. However, unclassified OTUs accounted for a higher relative abundance when using the ISPro method (35 %) compared to targeted metagenomics (5 %). The two methods performed comparably in terms of cost and time; however, the IS-Pro method was more user-friendly. CONCLUSIONS : It is essential to understand the value of different methods for characterisation of the microbiome. Targeted metagenomics and IS-Pro methods showed differences in ability in identifying and characterising OTUs, diversity and microbial composition of the lung microbiome. The IS-Pro method might miss relevant species and could inflate the abundance of Proteobacteria. However, the IS-Pro kit identified most of the important lung pathogens, such as Burkholderia and Pseudomonas and may work in a more diagnostics-orientated setting. Both methods were comparable in terms of cost and time; however, the IS-Pro method was easier to use.SUPPLEMENTARY MATERIAL: Table S1. Inclusion and exclusion criteria for COPD patients in this study. Table S2. Clinical characteristic of patients. Table S3. Comparison of the number of amplicons and operational taxonomic units for each sample for the targeted metagenomics and IS-Pro methods. Figure S1. Relative abundance of specific phyla in the sputum microbiome of COPD participants as detected by targeted metagenomics and IS-Pro methods (n = 23). The dots represent the different abundances of each sample, according to the different phyla. Phyla that are depicted with a single line on the y-axis were not present in any samples for that method. Figure S2. Bar plots showing the relative abundance of genera in the sputum microbiome of COPD participants as characterised by targeted metagenomics and IS-Pro methods (n = 23). The operational taxonomic units that could not be classified at a genus level are indicated as NA on the graph. Figure S3. The distribution of the unclassified operational taxonomic units (OTUs) at a class level of the sputum microbiome of COPD participants for targeted metagenomics and IS-Pro methods by phyla. At a class level, all the OTUs from targeted metagenomics could be classified.National Health Laboratory Service of South Africa (NHLS) Research Trusthttps://bmcmicrobiol.biomedcentral.comam2022Internal MedicineMedical Microbiolog

Expression of a Bacillus α-amylase gene in yeast

A recombinant plasmid, pSR11.3, containing the α-amylase gene (AMY) of Bacillus amyloliquefaciens was characterized and expressed in Bacillus subtilis. A 2.3 kilobase BamHI-BglII fragment carrying AMY was cloned into pBR322 (pEL322) and in both orientations into a multi-copy Escherichia coli-yeast shuttle vector YEp13 (pAM13) and expressed in E. coli HB101 and various Saccharomyces strains. We report on the successful secretion of an active bacterial enzyme in yeast without using yeast promoter and secretory signals. Enzyme production in B. subtilis 1A297(pSR11.3), E. coli HB101(pEL322) and Saccharomyces JM277315B(pAM13) transformants was measured as 125, 22 and 123 U/ml, respectively. The molecular weight of the purified α-amylase secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) was estimated to be 55 kDa. The pH and temperature optima for the α-amylase activities of the transformants were 6.5 to 8.0 and 50 to 65 °C, respectively. Amylose hydrolysis profiles of the α-amylases secreted by B. subtilis 1A297(pSR11.3) and Saccharomyces JM2773-15B(pAM13) indicate effective meso-thermostable hydrolytic enzymes with maltotriose and maltose, respectively, as major end products. © 1988 Springer-Verlag.Articl